Epilepsy is a widespread neurological disorder that affects approximately 50 million people worldwide (1). According to the World Health Organization (WHO), about 1% of the total burden of disease corresponds to various forms of epilepsy. Its pharmacologic treatment comprises a number of currently available antiepileptic drugs (AEDs) (2). The main problem concerning AEDs is the high incidence of side effects ranging from gastrointestinal distress, hepatotoxicity, depression, cognitive impairment and even refractory seizures (2) (3) (4) (5) (6). Moreover, about one third of patients suffering from epilepsy remain resistant to available treatments (1) (2). Hence, there is a clear need to continue to identify novel AEDs that control seizures with minimal adverse effects.
Medicinal plants and the chemical compounds contained therein represent a potential source of novel AEDs. Numerous studies on the use of ethnomedicinal plants for the treatment of seizures have been reported (7). Small molecule compounds and essential oils extracted from plants have been shown to exhibit anticonvulsant properties (18) (19) (20). One compound, losigamone, derived from the kava kava plant and originally used by traditional healers in the South Pacific as an anxiolytic, is now in early clinical development as a novel antiepileptic drug (8) (9). Another plant, Curcuma longa L., is a medicinal perennial herb of the Zingiberaceae family native to South Asia. It has been traditionally used as a carminative, laxative, anthelmintic and as a treatment for liver disorders. The powder of its rhizomes, turmeric, has been used not only as a condiment and color additive in food but also in traditional medicine against epilepsy (10). Its major active chemical constituents are the curcuminoids (3-5%) and the volatile turmeric oil (2-7%). Turmeric oil is mainly composed of bisabolene sesquiterpenoids: ar-, α-, β-, turmerone, α-atlantone and curlone, whereas the curcuminoids include curcumin, monodemethoxycurcumin and bisdemethoxycurcumin. Nearly all investigations on the medicinal properties of turmeric have been focused on curcumin, whose anticonvulsant activities have been demonstrated in several rodent models such as the iron-induced epileptogenesis (11), maximal electroshock (12), kainic acid-induced (13) and pentylenetetrazole-kindling (14) models. However, while a few studies on the neuroprotective activity of turmeric oil have been performed (15) (16) (17), a specific link between anticonvulsant activity and non-curcuminoid compounds such as volatile turmeric oil or bisabolene sesquiterpenoids has not been evaluated. Notably, previous studies on the volatile constituents of turmeric oil were limited due to the complex isolation steps involved.
Described herein is a practical method to isolate the main constituents of turmeric oil through RP-HPLC. The isolated compounds were individually evaluated in two vertebrate model systems: the zebrafish (Danio rerio) and the mouse (Mus musculus). Over the past decade, the zebrafish has emerged as a valuable model for genetic studies and drug screening. The strength of this in vivo model relies on its high genetic, physiologic and pharmacologic homology to humans. Their high fecundity and small size allow for the performance of tests in a medium- to high-throughput fashion using minute (microgram-scale) quantities of compound. The zebrafish also holds promise as an in vivo model for identifying novel neuroactive compounds since the dopaminergic, serotonergic, and GABAergic systems develop early during embryogenesis and are already functional in larvae (21). In addition, their rapid development ex utero and optical transparency makes it possible to easily detect morphological and behavioral effects of test compounds on living embryos and larvae (22).
More recently, zebrafish have also proven useful for the primary screening of potential novel anticonvulsants (23) (24) (25). An acute zebrafish seizure model based on the proconvulsant pentylenetetrazole (PTZ) has been described (23). The exposure of zebrafish larvae to PTZ evoked a sequence of behavioral changes, which were classified into three phases: a notable increase in swimming activity (stage I); rapid “whirlpool-like” circular swimming motion (stage II), and clonic movements with subsequent loss of posture and loss of movement for 1-3 seconds followed by tonic contractions (stage III) (23). In addition, electrophysiological recordings confirmed that zebrafish larval brains treated with PTZ displayed a series of ictal and interictal discharges. A follow-up study validated this zebrafish chemoconvulsant model by showing that 13 out of 14 clinically used AEDs were capable of suppressing PTZ-induced seizure behaviors in zebrafish (24).
In the course of screening a series of medicinal plants for their potential anticonvulsant activities in the zebrafish PTZ chemoconvulsant model, we confirmed the reported anticonvulsant properties of curcumin. Surprisingly, however, further testing of turmeric oil and its chromatographic fractions revealed additional constituents capable of suppressing PTZ-induced seizure behaviors in larval zebrafish. Mass spectrometry and NMR analysis of these active purified fractions revealed them to belong to the bisabolene sesquiterpenoids ar-turmerone, α-, β-turmerone (curlone) and α-atlantone. The anticonvulsant activities identified using the zebrafish PTZ assay were then confirmed in the equivalent mouse PTZ-induced seizure model and the 6 Hz psychomotor seizure model of partial epilepsy. Additionally, an assessment on motor coordination and balance was performed jn mice using the elevated bridge after i.v. injection of ar-turmerone in order to determine any side effects leading to motor impairment.
There have been some publication providing turmeric extracts, for use in medicine, however, none of them provides the use thereof as an anticonvulsant agent in the treatment of disorders of the central nervous system. For example WO2007109210 and WO2010045577 provide extracts of curcuma plants, and methods of treating neurodegenerative disorders such as disorders associated with amyloid plaque aggregation or fibril formation (e.g. Alzheimer's disease), however, neither patent application discloses or suggests a potential use of curcuma extracts as anticonvulsant agents. WO2011080090 provides formulations of turmeric oil having anti-inflammatory, analgesic and/or anti-cancer activities, however, again it neither discloses nor suggests a potential use of curcuma extracts as anticonvulsant agents.
Even more, it is known that bisabolene-type sesquiterpenoids exhibit Acetylcholine esterase inhibitory activity (41), whereas it has also been shown that AChE blockers, in general induce seizures and may lead to status epilepticus, resulting in spontaneous seizures following a latent period (42). It was therefore surprising to find that bisabolene-type sesquiterpenoids are in fact capable of reducing the extent of epileptic seizures, rendering them suitable as anticonvulsant agents in the treatment of central nervous system disorders.